We study how nutrient availability coordinates global patterns of bacterial gene expression as well as pathway-specific regulation. We focus on the role of two 3'-pyrophosphorylated analogs of GDP and GTP, called (p)ppGpp. Responses to nutrient limitation and adaptation involve fluctuations in levels of (p)ppGpp, whether starving for amino acids, phosphate, nitrogen or energy sources. Most (p)ppGpp regulation is thought to be directly on RNA polymerase initiation or indirectly on transcription modified by alternative sigma factors, such as RpoS, a master determinant of starvation-specific gene expression. This year we learned that induction of RpoS by (p)ppGpp occurs by changing the efficiency of translation of rpoS mRNA instead of transcriptional effects on mRNA abundance. We think this involves the dksA gene, since a dksA deletion blocks (p)ppGpp induction of RpoS and overexpression of DksA, in the absence of (p)ppGpp, induces RpoS. The translational effects of (p)ppGpp and DksA have been found exerted on a region of RpoS mRNA far upstream of the rpoS mRNA leader region where the SD sequence is known to be sequestered by RNA folding and regulated by other factors. DksA effects are puzzling: increased (p)ppGpp does not induce DksA nor does increased DksA induce (p)ppGpp. Another clue is our discovery that requirements for several amino acids due to a dksA deletion are the same as for a complete (p)ppGpp deficiency. Amino acid starvation by codon-specified uncharged tRNA binding is long known to induce synthesis of (p)ppGpp on the ribosome by the RelA protein but few details are available on the RelA-ribosome interaction. We have now mapped RelA differences in dimethylsulfate modification of rRNA by RelA biding. With solved ribosome structures as a guide, perturbations of rRNA structure on the interior surfaces of both the large and small ribosomal subunits can be localized. The DMS modifications accompanying RelA binding occur in the region of tRNA-CCA end binding as well as in the decoding region of A site bound tRNA.
